Use of agonists or antagonists of P2 purinoceptors for the prevention glutamate-evoked cytotoxicity

ABSTRACT

Compounds that are agonists of P2 purinoceptors, such as 5-adenylylimidodiphosphate, are used to treat conditions such as pain, hormonal imbalance, high blood pressure, thermoregulation, respiration, learning, patter recognition and memory.

BACKGROUND OF THE INVENTION

[0001] The present invention relates to the use of a specific class ofcompounds for the prevention of glutamate-evoked cytotoxicity.

STATE OF THE ART

[0002] Glutamate constitute the major excitatory neurotransmitter of thecentral nervous system (Hollmann M., Heinemann S., Annu. Rev. Neurosci.17, 31-108, 1994) and the ubiquitous distribution of glutamate receptorsthroughout the CNS proves that glutamate plays a central role in a widerange of physiological as well as pathological events (Watkins J. C.,Collingridge G. L., The NMDA receptor, IRL Oxford, 1989).

[0003] By most plausible theories and several experimental findings itis suggested a central role for glutamate-dependent neurotransmission infunctions such as learning, pattern recognition, and memory (Bliss T. V.P. Collingridge G. L., Nature 361, 31-39, 1993).

[0004] It has also been known for decades that glutamate is toxic toneurons in vivo and in culture and that glutamate receptor functioningis crucial in a number of brain diseases and injuries (Appel S. H.,Trends Neurosci. 16, 3-5, 1993). Many neurological illnesses involvingstrokes or epileptic seizures result in fact in brain damage justbecause of over-stimulation by glutamate, and degenerative diseasesamong which Alzheimer's, Huntington's, Parkinson's and amyotrophiclateral sclerosis (ALS) involve neuronal cell death caused by excessiveactivation of the glutamate receptors.

OBJECTS OF THE INVENTION

[0005] Object of the present invention is to provide a specific class ofcompounds for the modulation of glutamate-evoked neurotransmission andneurotoxicity, which allows the possible treatment of acute and chronicneurodegenerative diseases.

[0006] Another object of the present invention is to provide a specificclass of compounds which allows the modulation of glutamate-relatedphysiological functions, among which pain, hormonal balance, bloodpressure, thermoregulation, respiration, learning, pattern recognitionand memory.

[0007] Still an object of the present invention is to provide a specificclass of compounds which can be used as pharmacological tools for theprevention of glutamate-evoked cytotoxicity.

[0008] A further object of the present invention is to provide aspecific class of compounds which represents a valid pharmacologicalalternative to previously described compounds, such as competitive andnon-competitive glutamate antagonists, gangliosides and growth factors,for the treatment of acute and chronic glutamate-related neurologicaldiseases.

DESCRIPTION OF THE INVENTION

[0009] These and still other objects and related advantages which willbe more clearly stressed by the following description are achieved bythe use of compounds which are agonists or antagonists of P₂purinoceptors for the prevention of glutamate-evoked cytotoxicity.

[0010] The fundamental novelty of this invention is the correlationbetween glutamate-evokes biological events and P2 purinoceptormodulators (agonists or antagonists). Both glutamate receptors and P2purinoceptors share in fact the property of being ionotropic as well asmetabtropic kinds of receptors.

[0011] As an example, we have chosen the compounds Basilen Blue E 3G(also called Reactive Blue 2) and Cibacron Blue 3GA, which areantagonists of P₂ purinoceptors. These compounds can be purchased, forexample, from Sigma and their molecular structures and maincharacteristics are described on the 1995 Sigma catalog distributed inItaly, respectively on pages 149 for Basilen Blue E-3-G and 266 forCibacron Blue 3GA. The other compound we have chosen is5-adenylylimidodiphosphate (AMPPNP), which is an agonist of P2purinoceptors. This compound can be also purchased from Sigma and itsmolecular structure and main characteristics are described in page 52 ofthe Sigma catalog published in Italy in 1995.

[0012] Always according to the present invention, these compounds areused to prevent glutamate-evoked cytotoxicity in the nervous system'scells, particularly in CNS neurons. As a cellular model system for CNSneurons we have adopted postnatal rat cerebellar neurons. These cells,which are among the best characterized primary neuronal cultures, whenisolated from postnatal rat cerebellum (Lasher R. S., and Zagon I. S.,Brain Res. 41, 428-438, 1972), develop in vitro their mature phenotypeas interneurons which use glutamate as a neurotransmitter andfurthermore constitute an excellent model system for the study ofglutamate-mediated cytotoxicity.

[0013] By exposing granule neurons to 100 μM glutamate for 15-30 min isobtained (after 15-20 hours) 80-100% of total cell death. We find thatthe P₂ purinoceptor antagonist basilen blue, also called reactive blue 2(an anthraquinone sulfonic acid derivative), when somministered togranule neurons at 100 μM in the simultaneous presence of glutamate,completely sustains cellular survival, thereby abolishing the cytotoxicaction of glutamate. The effects of basilen blue on cerebellar granulecell morphology reveal, despite the exposure to glutamate, whichotherwise induces complete cell death, apparently healty-looking cellbodies that do bear a dense network of highly branching processes.Adhesion and neurites fasciculation are also preserved by basilen blue.The acute response characterized by rapid swelling of the cell body andloss of brightness, generally observed in granule neurons within thefirst 5 min of treatment with glutamate, is furthermore prevented byaddition of basilen blue, suggesting that the compound probably actsvery early in the chain of events immediately downstream of theEAA-receptor interaction.

[0014] It is important to stress that basilen blue per se, up to thehighest concentration tested of 300 μM, is not toxic to the cells and,when somministered to granule neurons for 0.5-26 hours, does not affectplasma membrane permeability (as measured by ethidium bromide uptake),or cellular metabolism (as measured by conversion of MTT into formazanby mitochondrial dehydrogenase activities). Basilen blue preventsglutamate-evoked cell death with an IC50 in the 10-20 μM range, valuegenerally in agreement with the concentrations of the compound reportedfor P2 purinoceptor antagonism. The other commercially available isomerof the sulfonic derivatives of anthraquinone (cibacron blue) iseffective in this regard. Caffeine, a P1 purinoceptor antagonist, up to100 μM does not abolish the cytotoxic action of glutamate.

[0015] The effects of basilen blue on protection from cytotoxicity arelinear with time and depend on the modality of somministration of thecompound. When basilen blue is added to the cells 10 min afterglutamate, and then incubated with granule neurons for only 15 min, itprotects from cell death 60-70% of the entire neuronal population; ifsomministered only for the last 5 min of treatment of the cells withglutamate, basilen blue sustains survival of 25-40% of total neurons.When instead it is added 1-2 min or 30 min or 2 hours after exposure ofgranule neurons to glutamate (and then incubated with the cells for thenext 20 hours) basilen blue protects from cell death respectively55-70%, 30% and 10% of the total neuronal population. When it is addedbefore (not during or after) exposure of granule neurons to glutamate,basilen blue requires a pretreatment of at least 20-25 hours in order toprevent by 70-80% the cytotoxic action of glutamate. Inhibition ofaspartate uptake is not obtained as a consequence of these sametreatments. Independently from the modality of somministration togranule neurons, the prevention of cytotoxicity induced by basilen bluedoes not depend on new protein synthesis, since it is insensitive toinhibitors such as actinomycin D (used at 10 μM) or anisomycin (used at100 μM).

[0016] Basilen blue inhibits binding of [³H] ATP to membranes of granuleneurons with an IC50 of about 10 μM, that corresponds to the IC50 thatprevents glutamate-evoked cytotoxicity. Binding studies with [³H] ATPhave been performed also directely with intact cells and basilen bluewas shown to be as effective.

[0017] We have cultured the cells in the continuous presence (from day 1but not later than day 2 in vitro) of 100 μM 5-adenylylimidodiphosphate(AMPPNP), a known P₂ purinoceptor agonist. Glutamate-inducedcytotoxicity is inhibited by approximately 50-60% as a consequence ofthis treatment. An acute exposure of the cells to 100 μM AMPPNP(simultaneously with glutamate) is not effective in this regard. Thefact that by culturing the neurons in the continous presence of AMPPNPelicits the same effect caused by an acute exposure to basilen bluesupports our hypothesis of a direct involvement of purinoceptors inglutamate-dependent neurotoxicity; it moreover suggests that aphenomenon of desensitization of purinoceptors most likely takes placein cerebellar granule cells.

[0018] Since D-[³H] aspartate release has been often used as a measureof the functional state of cerebellar granule neurons cultured in vitroand depolarization- or glutamate-evoked aspartate release is a featureprogressively aquired by these cells along with neuronal maturation, wedecided to test this parameter to further study the biological effectsand possible mechanisms used by basilen blue in the prevention of celldeath. We find that basilen blue inhibits glutamate-induced release of[³H] aspartate with an IC50 of about 10 μM. The inhibition is almostcomplete but does not affect basal release and it occurs when therelease is measured for 1 min, for longer times (3,10, and 25 min) oreven in the presence of Mg²⁺. Also a cronic exposure of granule neuronsfor 8 days to 100 μM AMPPNP inhibits by 70-80% the glutamate-evokedrelease of [³H] aspartate.

[0019] Glutamate-dependent neurotoxicity is often accompanied also incerebellar granule neurons with increase in intracellular Ca²⁺ via amultistep process. Basilen blue, differently from caffeine, almostcompletely abolishes glutamate-evoked, but not basal, Ca²⁺ uptake withan IC50 of approximately 10 μM. Also this value is in accordance withthe IC50 found for inhibition of ATP binding, cytotoxicity and aspartaterelease. The basilen blue-dependent inhibition occurs when Ca²⁺ uptakeis measured for short (1 min) or for longer times (3, 10, and 25 min).The cronic exposure of granule neurons for 8 days to 100 μM AMPPNPinhibits by 50-70% glutamate-evoked Ca²⁺ influx, similarly to theinhibition of cytotoxicity and aspartate release.

DESCRIPTION OF DRAWINGS

[0020] SHEETS ⅛ and {fraction (2/8)}

[0021] Basilen Blue prevents cytotoxicity induced by glutamate incerebellar granule primary cultures: effects of dose-response andmodality of addition. Replicate cerebellar granule cultures at 8 DIVwere exposed for 25 min to 100 μM glutamate, in the simultaneouspresence of different concentrations of basilen blue (FIG. 1). After 20hours, the cultures were assessed for cultures were assessed for cellsurvival by direct count of intact viable nuclei. The asteriskrepresents the % of nuclei obtained after simultaneous addition to thecells of 100 μM glutamate and 100 μM caffeine. In FIG. 2, replicatecerebellar granule cultures at 8 DIV were exposed for 25 min to 100 μMglutamate. At different time after glutamate withdrawl, basilen blue(100 μM) was added to the medium until the cultures, 20 hours later,were assessed for cell survival by direct count of intact viable nuclei.The asterisk represents the % of nuclei obtained after simultaneousaddition to the cells of 100 μM glutamate and 100 μM basilen blue. InFIG. 3, replicate cultures were pretreated in the presence of 100 μMbasilen blue for different time, before the addition of 100 μM glutamatefor 25 min (performed in the absence of basilen blue). After 20 hoursthe cultures were assessed for cell survival.

[0022] The asterisk represents the % of nuclei obtained aftersimultaneous addition to the cells of 100 μM glutamate and 100 μMbasilen blue. Counts represent means±SEM (n=4) and 100% of cell survivalrepresents 1.75−2×106 total cells.

[0023] METHODS. Approximately 20 hours after exposure of granule cellsto glutamate, the culture medium was removed and replaced with 1 ml of adetergent-containing lysing solution (0.5% ethyl hexadecyldimethyl—ammonium bromide, 0.28% acetic acid, 0.5% Triton X-100, 3 mMNaCl, 2 mM MgCl₂, in PBS pH 7.4 diluted {fraction (1/10)}).

[0024] After 1-2 minutes, the cells were triturated a few times,providing a uniform uspension of single, intact viable nuclei. Thelatter were quantified by ounting in a hemacytometer. Broken or damagednuclei were not included in the counts.

[0025] SHEETS ⅜ and {fraction (4/8)}

[0026] Basilen Blue inhibits binding of ATP to cerebellar granule cellmembranes, but to uptake of aspartate. Membranes were prepared from 8DIV cerebellar granule ells and 20 μg of protein were incubated with[³H] ATP (0.5 μCi/ml, final concentration 14 mM) (FIG. 4), in thepresence of different concentrations of basilen blue, for 1 h at 4° C.Specific binding is shown and counts represent means±SEM (n=3). Theasterisk represents the binding performed in the presence of 100 μMcaffeine. In FIG. 5, replicate cerebellar granule cultures at 8 DIV werewashed wice and incubated for different times in Locke's solution with[³H] D-2,3 aspartic acid (1 μCi/ml, final concentration 40 nM), in thepresence of different concentrations of basilen blue. After two washes,cells were dissolved in 0.1 M NaOH and counted for incorporatedradioactivity by liquid scintillation counting. Values are shown ascpm/μg and represent means±SEM (n=3). Protein concentrations weredetermined by the method of Bradford with ovine serum albumin asstandard.

[0027] METHODS. Replicate cerebellar granule cells at 8 DIV werecollected in ice-cold buffer A (50 mM Tris, 1 mM EGTA, adjusted to pH7.4 with HCl, containing also 2 mM phenylmethylsulfonyl fluoride, 200KTU/ml of aprotinin and 1 μg/ml of leupeptin) and were centrifuged at35.000×g for 20 min at 4° C. The pellet was resuspended in buffer A togive a protein concentration of 5-6 mg/ml and used immediately forbinding studies. After binding with [³H] ATP, the samples (1 ml) werevacuum-filtered through Whatman GF/B glass fiber filters and the filterswere immediately washed (3×4 s) with 5 ml of 50 mM Tris-HCl (pH 7.4),air dried and assessed for specifically bound radioactivity by liquidscintillation counting.

[0028] SHEETS ⅝ and {fraction (6/8)}

[0029] Culturing cerebellar granule cells in the presence of AMPPNP:modulation of glutamate-dependent cell death, Ca⁺⁺ uptake and aspartaterelease. Primary cerebellar granule cultures were prepared and startingat 1 DIV some of time were daily supplemented with 100 μM AMPPNP.(FIG.6) At 8 DIV, after two washes, some of the cultures were incubated at20° C. for 25 min with 100 μM glutamate. The following day, they wereassessed for cell survival, as previously described.

[0030] (FIG. 7) Replicate cerebellar granule cultures at 8 DIV wereincubated for 1 min in Locke's solution in the presence of 45 Ca⁺⁺ (1μCi/ml), with or without 100 μM glutamate, and were then assessed forCa⁺⁺ influx. (FIG. 8) Replicate cerebellar granule cultures at 8 DIVwere incubated for 5 min in Locke's solution, in the presence of [³H]D-2,3 aspartic acid (1 μCi/ml, final concentration 40 nM) and were thenassessed for aspartate release, in the presence or absence of 100 μMglutamate. Data are expressed as means±SEM (n=3).

[0031] SHEETS ⅞ and {fraction (8/8)}

[0032] Basilen Blue inhibits release of aspartate and uptake of Ca⁺⁺induced by glutamate in cerebellar granule cells. (FIG. 9) Replicatecerebellar granule cultures at 8 DIV were incubated for 5 min in Locke'ssolution in the presence of [³H] D-2,3 aspartic acid (1 μCi/ml, finalconcentration 40 nM). After two washes, the cultures were then incubatedfor 1 min in Locke's solution with or without 100 μM glutamate and inthe presence of different concentrations of basilen blue. The bufferremoved from the cultures during this release phase was collected intovials and counted for radioactivity. The asterisk represents the releaseof aspartate obtained in the simultaneous presence of 100 μM glutamateand 100 μM caffeine. Cells were dissolved in 0.1 M NaOH and proteinconcentrations were determined by the method of Bradford, with bovineserum albumin as standard. (FIG. 10) Replicate cerebellar granulecultures at 8 DIV were incubated for 1 min in Locke's solution in thepresence of 45 Ca⁺⁺ (1 μCi/ml), with our without 100 μM glutamate, andin the simultaneous presence of different concentrations of basilenblue. After two washes with ice-cold 154 mM choline chloride, 2 mM EDTA,the cells were lysed in 0.1 M NaOH and aliquots were collected formeasuring Ca⁺⁺ influx (by radioactive counting) and proteinconcentration. The asterisk represents the Ca⁺⁺ influx obtained in thesimultaneous presence of 100 μM glutamate and 100 μM caffeine. Data areexpressed as means±SEM (n=3).

1. Compounds agonists of P2 purinoceptors for the prevention ofglutamate-evoked cytotoxicity.
 2. Pharmacological compositioncomprising, as active ingredients, compounds agonists of P₂purinoceptors, in addition to usual additives.
 3. Pharmacologicalcomposition according to claim 2, which comprises compounds agonists ofP₂ purinoceptors as active agent, in addition to usual additives, in apharmacologically acceptable means.
 4. Pharmacological compositionaccording to claim 2, wherein said agonists of P₂ purinoceptors are5-adenylylimidodiphosphate (AMPPNP).
 5. Method for the modulation ofglutamate-evoked phisiological functions in a mammal in need thereof,which comprises administering to said mammal a pharmacologicalcomposition according to claim
 2. 6. Method according to claim 5,wherein said glutamate-evoked physiological functions are pain, hormonalbalance, blood pressure, thermoregulation, respiration, learning,pattern recognition and memory.
 7. Method for the treatment of pain,hormonal balance, blood pressure, thermoregulation, respiration,learning, pattern recognition and memory in a mammal in need thereof,which comprises administering to said mammal a pharmacologicalcomposition according to claim
 2. 8. Method for the treatment of acuteand chronic neurodegenerative diseases in a mammal in need thereof,which comprises administering to said mammal a pharmacologicalcomposition according to claim
 2. 9. Method for the prevention ofglutamate-evoked neurotoxicity which comprises administering apharmacological composition according to claim
 2. 10. Method for thetreatment of pain, hormonal balance, blood pressure, thermoregulation,respiration, learning, pattern recognition and memory in a mammal inneed thereof, which comprises administering to said mammal an effectiveamount of 5-adenylylimidodiphosphate (AMPPNP).